Project Abstract Advanced cell-based technologies for kinase activity profiling are needed for earlier identification of next- generation kinase inhibitors and to enable clinical evaluation of kinase activity biomarkers. The long term goal of this proposal is to generate targeted kinase activity tests compatible with viable cells from patient-derived samples. The objectives in this proposal work toward that goal by developing prototype assays that measure Jak2 and Src-family (SFK) kinase activity specifically in disease-relevant cellular models. Peptide-based substrates phosphorylated specifically by Jak2 and SFK have been validated in vitro. In this proposal they will be adapted for cell-based use, and it will be shown that intracellular phosphorylation of the substrates is specifically driven by the kinase of interest. In addition, the assays will be analytically characterized and used in screening applications relevant to drug discovery. Successful completion of these prototype assays will validate an R&D pipeline for developing additional cell-based targeted kinase activity assays. In Phase II, cell-based assays for additional kinases will be developed, partnerships for commercialization in the drug discovery kinase profiling market will be formalized, and compatibility with viable cells derived from patient samples will be evaluated.